This invention relates to the manufacturing of supports having bound to the surfaces thereof a plurality of chemical compounds such as polymers, which are prepared on the surface in a series of steps. More particularly, the present invention relates to methods for solid phase chemical synthesis, particularly solid phase synthesis of oligomer arrays, or attachment of oligonucleotides and polynucleotides to surfaces, e.g., arrays of polynucleotides.
In the field of diagnostics and therapeutics, it is often useful to attach species to a surface. One important application is in solid phase chemical synthesis wherein initial derivatization of a substrate surface enables synthesis of polymers such as oligonucleotides and peptides on the substrate itself. Support bound oligomer arrays, particularly oligonucleotide arrays, may be used in screening studies for determination of binding affinity. Modification of surfaces for use in chemical synthesis has been described. See, for example, U.S. Pat. No. 5,624,711 (Sundberg), U.S. Pat. No. 5,266,222 (Willis) and U.S. Pat. No. 5,137,765 (Farnsworth).
Determining the nucleotide sequences and expression levels of nucleic acids (DNA and RNA) is critical to understanding the function and control of genes and their relationship, for example, to disease discovery and disease management. Analysis of genetic information plays a crucial role in biological experimentation. This has become especially true with regard to studies directed at understanding the fundamental genetic and environmental factors associated with disease and the effects of potential therapeutic agents on the cell. Such a determination permits the early detection of infectious organisms such as bacteria, viruses, etc.; genetic diseases such as sickle cell anemia; and various cancers. This paradigm shift has lead to an increasing need within the life science industries for more sensitive, more accurate and higher-throughput technologies for performing analysis on genetic material obtained from a variety of biological sources.
Unique or misexpressed nucleotide sequences in a polynucleotide can be detected by hybridization with a nucleotide multimer, or oligonucleotide, probe. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. These techniques rely upon the inherent ability of nucleic acids to form duplexes via hydrogen bonding according to Watson-Crick base-pairing rules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. An oligonucleotide probe employed in the detection is selected with a nucleotide sequence complementary, usually exactly complementary, to the nucleotide sequence in the target nucleic acid. Following hybridization of the probe with the target nucleic acid, any oligonucleotide probe/nucleic acid hybrids that have formed are typically separated from unhybridized probe. The amount of oligonucleotide probe in either of the two separated media is then tested to provide a qualitative or quantitative measurement of the amount of target nucleic acid originally present.
Direct detection of labeled target nucleic acid hybridized to surface-bound polynucleotide probes is particularly advantageous if the surface contains a mosaic of different probes that are individually localized to discrete, known areas of the surface. Such ordered arrays containing a large number of oligonucleotide probes have been developed as tools for high throughput analyses of genotype and gene expression. Oligonucleotides synthesized on a solid support recognize uniquely complementary nucleic acids by hybridization, and arrays can be designed to define specific target sequences, analyze gene expression patterns or identify specific allelic variations. The arrays may be used for conducting cell study, for diagnosing disease, identifying gene expression, monitoring drug response, determination of viral load, identifying genetic polymorphisms, analyze gene expression patterns or identify specific allelic variations, and the like.
In one approach, cell matter is lysed, to release its DNA as fragments, which are then separated out by electrophoresis or other means, and then tagged with a fluorescent or other label. The resulting DNA mix is exposed to an array of oligonucleotide probes, whereupon selective binding to matching probe sites takes place. The array is then washed and interrogated to determine the extent of hybridization reactions. In one approach the array is imaged so as to reveal for analysis and interpretation the sites where binding has occurred. Arrays of different chemical probe species provide methods of highly parallel detection, and hence improved speed and efficiency, in assays. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
The arrays may be microarrays created by in-situ synthesis, oligonucleotide deposition or cDNA. In general, arrays are synthesized on a surface of a substrate by one of any number of synthetic techniques that are known in the art. In one approach to the synthesis of microarrays flow cells or flow devices are employed in which a substrate is placed to carry out the synthesis.
One embodiment of the present invention is a method for synthesizing a plurality of chemical compounds on the surface of a support wherein the synthesis comprises a plurality of steps. The method comprises performing at least two of the steps by placing a support having a functionalized surface into a chamber of one flow cell, arbitrarily designated as a first flow cell, and subjecting the surface to a step, arbitrarily designated as a first step, of the synthesis and placing the support into a chamber of another flow cell, arbitrarily designated as a second flow cell, and subjecting the surface to another step, arbitrarily designated as a second step, of the synthesis.
Another embodiment of the present invention is a method for synthesizing a plurality of biopolymers on the surface of a support wherein the synthesis comprises a plurality of monomer additions. Each of the following steps is performed after each of the monomer additions. As above, the designations of first and second is arbitrarily applied to the flow cells and the steps involved. The support is placed into a chamber of a first flow cell and the surface thereof is subjected to a first step of the synthesis that is subsequent to a monomer addition. Then, the support is placed into a chamber of a second flow cell and the surface thereof is subjected to a second step of the synthesis that is subsequent to the first step. The steps are usually repetitive steps such as, for example, washing the surface of the surface, oxidizing substances on the surface of the support, removing protective groups from the surface of the support, and so forth.
Another embodiment of the present invention is an apparatus for synthesizing an array of biopolymers on the surface of a support. The apparatus comprises a plurality of flow cells. One or more fluid dispensing stations are in fluid communication with one or more of the plurality of flow cells. A station is included for monomer addition to the surface of the support. The apparatus further comprises a mechanism for moving a support to and from the station for monomer addition and a flow cell and from one flow cell to another flow cell.
Another embodiment of the present invention is an apparatus for synthesizing an array of biopolymers on the surface of a support. The apparatus comprises a plurality of flow cells, which may be mounted on the platform or other suitable frame. The flow cells comprise a chamber, a holder for the support, at least one inlet and an outlet, wherein each of the inlets is in fluid communication with a manifold. The outlet is in controlled fluid communication with one or more purification systems, holding chambers and sensors. One or more fluid dispensing stations are mounted on the platform and are in fluid communication with one or more of the plurality of flow cells by means of the manifolds. A station for monomer addition to the surface of the support is mounted on the platform. The apparatus also comprises a mechanism for moving a support to and from the station for monomer addition and a flow cell and from one flow cell to another flow cell. Also included is a controller for controlling the movement of the mechanism.